1. Field of the Invention
The present invention relates generally to transgenic plants. More specifically, it relates to transgene expression at a specific stage of plant development. Even more specifically the invention relates to novel Rubisco promoters of Brassica species, and DNA constructs or expression cassettes comprising at least one of the promoters for transformation of homologous or heterologous plants for efficient production of gene products, particularly for contained use.
2. Description of Related Art
Assimilation and conversion of atmospheric carbon dioxide via the reaction with ribulose-1,5-bisphosphate into phosphoglycerate strictly depends on the activity of Rubisco enzyme. Structurally it consists of eight small subunits (SSU) and eight large subunits (LSU). The SSU proteins are encoded by several genes located in plant nuclear genome, while LSU genes are found in plastid genome. The number of Rubisco SSU genes in different plants varies from four copies up to fifteen copies or more in some polyploid genomes. There are at least four copies of Rubisco SSU genes in Arabidobsis thaliana and twelve or even more copies in wheat.
Based on their structure and function these nuclear genes may form multigenic families. The structure of these families is extensively studied, for example, in Arabidopsis and tomato plants. In tomato there are five Rubisco SSU (rbcS) genes located in three chromosomal loci, one of these genes being situated in chromosome 3, and the other four in chromosome 2. Moreover, three of the genes are known to be organized in tandem array within a 10 kb region. The same situation is known to occur in Arabidopsis thaliana rbcS gene family.
In Brassica napus coding sequences, 5′ and 3′ regulatory regions of three rbcS genes have been cloned and sequenced (accession numbers X75334, X55937, X61097). It has been suggested that Brassica napus contains no more than three rbcS genes.
There are also cDNA sequences obtained from mRNA of Brassica napus rbcS genes (one of them has been published with accession number X07367).
There have also been attempts to clarify the fine structure of the promoters of Brassica napus rbcS genes. Essential regulatory elements, like TATA, G-, GS, and I-boxes, necessary for basic activity and light regulation of the promoters has been described, and also putative silencer elements in one of the promoters has been studied.
Clearly, however the information available on gene structure and activity does not enable identification of differently expressing members of Brassica rbcS gene family in different plant tissues or development stages and under various environmental conditions.
Transgenic plants are used increasingly for production of various desired proteins and other gene products. An important aspect in designing transgenic plants is how to obtain significant levels of transgene expression in desired plant tissues or at desired plant development phases. The role of promoters is essentially important in this aspect and there is a clear need for new plant promoters.
Outchkourov et al. (2002) cloned an abundantly transcribed rbcS1 of the Rubisco small-subunit gene family of Chrysanthemum species (Chrysantemum morifolium Ramat.). Outchkourov et al. showed that tobacco plants transformed with a gene cassette containing uidA gene under the control of rbsS1-promoter provided β-glucuronidase (GUS) levels up to 10% of total soluble proteins in the leaves.
Even if the Chrysanthemum Rubisco promoter cloned by Outchkourov et al. gives high protein expression levels in tobacco leaves it may not fit for purposes where protein production is needed at a specific stage of development, such as protein production in seedlings or in germinating seeds.
Plant seeds and cotyledons are particularly advantageous for production because at early cotyledon development, nutritional sources from seeds, including amino acids and oils are abundantly available as raw material for de novo protein synthesis. Additionally, the recovery of the expressed gene products from homogenized sprouts is easier and more efficient than from harvested leaves. Production of transgenic proteins in germinating seeds is an approach that can be realized in contained manner in a suitable laboratory. For such purposes a promoter being active during seed germination or cotyledon development is essential and the published rbcS promoters are not applicable.
For purposes of producing transgenic expression products in developing sprouts there is a need for promoters expressing strongly at late stages of cotyledon development, because then the leaf size of cotyledons is bigger than at early cotyledon development and the material needed for compound collection is easier and more efficient to harvest.
Moreover, another important prerequisite for foreign protein production in plant tissues is high expression level of the proteins, and therefore there is a clear need for new promoters giving high protein content at a specific development stage and/or in a specific organ of a plant.
A promoter, which is active during seed germination or cotyledon development, is of particular importance in the production of transgene products in contained conditions in a suitable laboratory. None of the rbcS promoters so far published are however applicable for said purpose in Brassicaceae plants. New promoters for different new applications are therefore clearly needed.